This invention is in the field of immunology. Specifically, the invention relates to the generation of human monoclonal antibodies, especially those that are specific for surface antigens representative of a particular cell type. The compositions and methods embodied in the present invention are particularly useful for generating human monoclonal antibodies that are of major diagnostic and/or therapeutic potential.
The discovery by Kohler and Milstein of murine hybridomas capable of secreting specific monoclonal antibodies against predetermined antigens ushered a new era in the field of clinical immunology. The clonal selection and immortality of such hybridoma cell lines assure the monoclonality, monospecificity and permanent availability of their antibodies. However, murine antibodies have severe limitations in clinical applications for humans because of their intrinsic immunogenicity which often leads to undesirable immune responses. For instance, when immunocompetent human patients are administered therapeutic doses of mouse monoclonal antibodies, the patients produce antibodies against the mouse immunoglobulin molecules; these human anti-mouse antibodies neutralize the therapeutic antibodies and can cause acute toxicity. Hence, it would be desirable to generate antibodies that have no such drawbacks.
Generation of human monoclonal antibodies has been practically difficult for a number of reasons. First, it is not practical to immunize a human being with an immunogen of interest. The human antibodies which have been produced have been based on the adventitious presence of an available spleen. While four alternative ways of generating human monoclonal antibodies with desired antigen-binding specificity have been developed, they also have pronounced disadvantages. The first approach involves the use of recombinant DNA technology to generate a chimeric antibody. Such antibody is produced by fusing the constant regions of the heavy and light chains of a human immunoglobulin with the variable regions of the non-human antibody that confirm the antigen-binding specificity. While the resulting chimeric partly xenogeneic antibody is substantially more useful than using a fully xenogeneic antibody, it still has a number of disadvantages. The identification, isolation and joining of the variable and constant regions require substantial work. In addition, the joining of a constant region from one species to a variable region from another species may change the specificity and affinity of the variable regions, so as to lose the desired properties of the variable region. Furthermore, there are framework and hypervariable sequences specific for a species in the variable region. These framework and hypervariable sequences may result in undesirable antigenic responses. A variation of this approach is to replace residues outside the antigen-binding domains of a non-human antibody with the corresponding human sequences (WO 94/11509). Such a process is again labor intensive.
The second approach for production of human monoclonal antibodies is the use of xenogenic mice as described in U.S. Pat. No. 5,814,318 and U.S. Pat. No. 5,939,598. These genetically engineered mice are capable of expressing certain unrearranged human heavy and light chain immunoglobulin genes, with their endogenous immunoglobulin genes being inactivated. Although the xe2x80x9cxenomousexe2x80x9d represents an alternative system for generating human antibodies, it does not necessarily fully mimic the human immune system: first, the entire repertoire of immunoglobulins has not been duplicated in the mice; second, the incomplete inactivation of endogenous immunoglobulin genes may result in chimeric antibodies; third, the identification of the desired monoclonal antibodies generally involves conventional hybridoma techniques, which in turn requires extensive screening and subcloning a large number of hybridomas in order to identify the desired antibodies.
The third approach of producing human monoclonal antibodies is phage display library construction. The process proceeds with extraction of mRNA from a repertoire of human peripheral blood cells, followed by construction of a cDNA library comprising sequences of the variable regions of preferably all immunoglobulins. The cDNAs are then inserted into phages to which to display the immunoglobulin variable region as Fab fragments. Theoretically, if the phage library is large enough, it is possible to isolate the particular phage displaying the desired Fab fragment by panning the phages against the antigen of interest. However, this method is generally applicable only to substantially purified antigens, and not to a mixture of antigens such as thousands of those surface antigens expressed on the cell.
Finally, immortalized human B cells have been employed for monoclonal antibody production. This approach involve the steps of: (a) isolation of peripheral blood lymphocytes enriched in B cells; (b) transformation of the B cells with EBV-viruses or fusion with immortalized human lymphoblastoid cells, followed by massive screening for the B cell transformants or hybridomas exhibiting the desired antigen-binding specificity. B cell transformation itself is an inefficient process yielding at best 0.1-10% stable transformants, thus most B cells with the desired specificity are lost in the pool used for subsequent selection process. Whereas researchers (e,g, Abe, Tsutomu et al. in EP 0218158) have attempted to enrich the population of B cells expressing the desired immunoglobulin by in vitro immunization/activation with the antigen of interest, the activation is again inefficient in the sense that non-specific B cells also proliferate during this process. The identification of specific B cells thus largely depends on the final stage of screening, during which tens and thousands of transformed B cell clones are tested for their abilities to bind the antigen. Like the aforementioned methods, this approach is time consuming, labor intensive, and not amenable to high throughput antibody screening and production.
Thus, there remains a considerable need for alternative routes for generating human monoclonal antibodies.
A principal aspect of the present invention is the design of an efficient screening technique applicable for the generation of human monoclonal antibodies. This technique allows production of specific antibodies with minimal labor. It is particularly useful for large-scale production of populations of human monoclonal antibodies that are immunoreactive with antigens representative of a particular cell type. Distinguished from the above-mentioned approaches that involve generation of hybridomas and/or immortalized B cells, the methods of this invention employ non-transformed B cells, and generate expanded non-transformed B cell clones with required antigen-binding specificity for antibody production and/or isolation of the genes encoding the antibody.
Specifically, the present invention provides a method for generating human monoclonal antibodies immunoreactive with a desired antigen, the method comprising: (a) providing an isolated population of human lymphocytes comprising a plurality of non-transformed human B lymphocytes; (b) selecting the non-transformed human B lymphocytes that specifically bind to the desired antigen; (c) culturing the B lymphocytes of (b) under conditions favorable for B cell proliferation to yield a plurality of isolated non-transformed B cell clones producing the human monoclonal antibodies immunoreactive with the desired antigen, and d) optionally isolating the non-transformed B cell clones.
In one aspect, selecting the non-transformed human B lymphocytes comprises contacting the population of human lymphocytes with the desired antigen under conditions favorable for specific binding of B lymphocytes to the desired antigen, and separating unbound B lymphocytes from B lymphocytes bound to the desired antigen.
In another aspect, the method outlined above further comprises: (e) isolating a polynucleotide comprising sequences encoding an antigen-binding fragment of the heavy chain of the human monoclonal antibody from the isolated non-transformed B cell clone of (d); (f) isolating a polynucleotide comprising sequences encoding an antigen-binding fragment of the light chain of the human monoclonal antibody from the isolated non-transformed B cell clone of (d); and (g) expressing the polynucleotides of (e) and (f) to yield the human monoclonal antibody or an antigen binding fragment thereof.
The isolation of the polynucleotide encoding the heavy chain may precede, or carry out subsequent to, or concurrently with the isolation of the polynucleotide encoding the light chain.
In a separate aspect, the antigen binding fragment encoded by the polynucleotides of the invention may be selected from the group consisting of bispecific antibodies, chimeric antibodies, Fab, F(abxe2x80x2)2, single chain V region fragments (scFv) and fusion polypeptides, wherein the fusion polypeptide comprises the antigen binding fragment conjugated to a chemically functional moiety. Within this embodiment, the term xe2x80x9cmoietyxe2x80x9d encompasses signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioresponse modifiers, toxins, detectable labels, and drugs.
In yet another separate aspect, the antigen employed in the antibody generation process is a biological or chemical compound. The antigen may be a cellular protein, such as a receptor ligand, secreted protein, cell surface receptor, cytosolic protein, and nuclear protein. In a preferred embodiment, the antigen is a surface antigen presented by an intact cell. Where an intact cell is employed for antigen presentation, the cell is preferably a eukaryotic cell, whose surface is free of serum.
In still yet another aspect, the polynucleotides encoding the immunoglobulins or fragments thereof are expressed by one or more gene delivery vehicle in a host cell. Suitable gene delivery vehicles include viral vectors, liposomes, and plasmids. The host cell is preferably a eukaryotic cell capable of performing post-translational modifications of immunoglobulins or their fragments.
The invention also provides a method of generating a population of human monoclonal antibodies that specifically bind to antigens representative of a specific cell type. The method involves: (a) providing an isolated population of human lymphocytes comprising a plurality of non-transformed human B lymphocytes; (b) selecting the non-transformed human B lymphocytes that specifically bind to cells of the specific type; and (c) culturing the B lymphocytes of (b) under conditions favorable for B cell proliferation to yield a plurality of non-transformed B cell clones, thereby generating a population of human monoclonal antibodies exhibiting binding specificity to antigens representative of the specific cell type.
In one aspect of this embodiment, the method further comprises: (d) isolating a population of polynucleotides comprising sequences encoding the antigen-binding fragments of the heavy or light chains of the population of human monoclonal antibodies from the plurality of non-transformed B cell clones; and (e) expressing the polynucleotides to yield a population of polypeptides of the human monoclonal antibodies or the antigen-binding fragments thereof.
Further provided is the isolated population of polynucleotide and the polypetides encoded thereby. In a preferred embodiment, at least one polypeptide of the population is conjugated to a chemically functional moiety. A preferred moiety is selected from the group consisting of signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioresponse modifiers, toxins, detectable labels, or drugs.
Also embodied in this invention is a population of human monoclonal antibodies generated by any of the invention methods disclosed herein.